Tissue-engineered gut-sphincter complexes and methods of making the same

ABSTRACT

Methods are disclosed for forming tissue engineered, tubular gut-sphincter complexes from intestinal circular smooth muscle cells, sphincteric smooth muscle cells and enteric neural progenitor cells. The intestinal smooth muscle cells and neural progenitor cells can be seeded on a mold with a surface texture that induces longitudinal alignment of the intestinal smooth muscle cells and co-cultured until an innervated aligned smooth muscle sheet is obtained. The innervated smooth muscle sheet can then be wrapped around a tubular scaffold to form an intestinal tissue construct. Additionally, the sphincteric smooth muscle cells and additional enteric neural progenitor cells can be mixed in a biocompatiable gel solution, and the gel and admixed cells applied to a mold having a central post such that the sphinteric smooth muscle and neural progenitor cells can be cultured to form an innervated sphincter construct around the mold post. This innervated sphincter construct can also be transferred to the tubular scaffold such that the intestinal tissue construct and sphincter construct contact each other, and the resulting combined sphincter and intestinal tissue constructs can be further cultured about the scaffold until a unified tubular gut-sphincter complex is obtained.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/066,492, filed Jun. 27, 2018, which is a 35 U.S.C. 371 national stagefiling of International Application No. PCT/US2016/068891, filed Dec.28, 2016, which claims priority to U.S. Provisional Application No.62/273,161, filed Dec. 30, 2015. The present application is also acontinuation-in-part of U.S. application Ser. No. 15/976,569, filed May10, 2018, which is a divisional application of U.S. application Ser. No.14/375,812, filed Jul. 31, 2014, now U.S. Pat. No. 9,993,505 issued Jun.12, 2018, which is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/2013/024080, filed Jan. 31, 2013,which claims priority to U.S. Provisional Application No. 61/592,890,filed Jan. 31, 2012 and U.S. Provisional Application No. 61/592,871,filed Jan. 31, 2012. The contents of the aforementioned applications arehereby incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with government support from National Institutesof Diabetes, Digestive and Kidney Diseases under NIH/NIDDK Grant No.RO1DK071614. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention concerns bioengineering of tubular tissuestructures, such as gastrointestinal tissues and, in particular,tissue-engineered gut-sphincter complexes.

BACKGROUND

The gastrointestinal (GI) tract or “gut” is a structurally complexhollow organ that displays diverse motility patterns to perform avariety of functions that aid in ingestion, digestion, absorption ofnutritive elements, and excretion of waste. Gastrointestinal motility isa result of chemical and electrical interactions between smooth muscle,intramural innervation, interstitial cells, and mucosal epitheliallayers.

Phasic neuromuscular structures of the GI tract contain orthogonallayers of smooth muscle, interlaced with enteric neuronal plexuses. Theyare also associated with interstitial cells and specialized mucosallayers. The propagating peristaltic wave defines the phasic nature ofthis neuromusculature. It encompasses contraction and relaxation of boththe circular and longitudinal smooth muscle layers. The neuronalcomponents as well as the interstitial cells generate electricalactivity for the coordination of peristalsis. This activity is coupledin a highly coordinated manner to intracellular biochemical events inthe smooth muscle layers to result in gut motility.

The principal components of the GI tract are the small intestine, thecolon and the anal sphincter. The small intestine is the primarynutrient absorptive structure of the GI tract. Peristalsis and segmentalcontractions of the small intestine increase the surface area tofacilitate greater absorption by the villi of the intestinal epithelium.The colon is contiguous with the small intestine, facilitating waterabsorption, and excretion of stool. The internal anal sphincter (IAS)contributes to 70% of the anal canal closure pressure, maintainingcontinence. Weakened mechanical efficiency of the IAS due to idiopathicsphincteric degeneration, surgical, or obstetric trauma all lead topassive and active incontinence.

In addition to the anal sphincter, the gastrointestinal system hasseveral other sphincters that control the transit of fluids through thegut, including the lower esophageal sphincter (LES) and the pyloricsphincter at the exit of the stomach.

In the basal state, the smooth muscle of the sphincters remains in astate of tonic contraction and closure to serve as a one-way valve toregulate flow through the opening controlled by the sphincter. Skeletalmuscle sphincters are under voluntary control while smooth musclesphincters are controlled by complex interactions between extrinsicnerves from the central nervous system (CNS) and intrinsic control bythe enteric nervous system and the myogenic properties of specializedsmooth muscle cells.

Sphincteric smooth muscles represent tonic muscles that remaincontracted at rest and have small amplitude, slow contraction and slowrelaxation response, while non-sphincteric smooth muscle representphasic muscle that shows a wide range of contractile activity varyingfrom a fully relaxed basal state to a large-amplitude rapid contractionand rapid relaxation response (Goyal et al., The GastrointestinalSystem, Motility and Circulation, in Handbook of Physiology, J. D. Woodand S. G. Schultz, Editors. 1989, The American Physiological Society:Bethesda. p. 865-908).

There exists a need for functional tissue-engineered sphinctericconstructs for repair and/or reconstruction of damaged sphincters aswell as tissue engineered gut-sphincter complexes for gut-lengtheningand other therapeutic interventions. Alternatively, such constructscould provide functional in vitro models of sphincters and gut segmentsfor development of therapies and/or drug testing.

SUMMARY

Methods and constructs are disclosed for bioengineering ofgastrointestinal tissue. In particular, three-dimensional,bioengineered, tubular gut-sphincter complexes are disclosed, togetherwith methods of forming such constructs.

In one aspect, methods of forming tissue engineered, tubulargut-sphincter complexes are disclosed that start by obtaining intestinalcircular smooth muscle cells from an intestinal donor source, obtainingsphincteric smooth muscle cells from a sphincteric donor source, andobtaining enteric neural progenitor cells from at least one neuralprogenitor donor source. The intestinal smooth muscle cells can beseeded on a mold with a surface texture that induces longitudinalalignment of the intestinal smooth muscle cells with the neuralprogenitor cells added to the intestinal smooth muscle cells on themold, such that the combination of intestinal smooth muscle cells andthe neural progenitor cells can be cultured until an innervated alignedsmooth muscle sheet is obtained. The innervated smooth muscle sheet canthen be wrapped around a tubular scaffold to form an intestinal tissueconstruct. Additionally, the sphincteric smooth muscle cells andadditional enteric neural progenitor cells can be mixed in abiocompatiable gel solution, and the gel and admixed cells can beapplied to a mold having a central post; the sphinteric smooth muscleand neural progenitor cells can be cultured to form an innervatedsphincter construct around the mold post. Once formed, the innervatedsphincter construct can also be transferred to the tubular scaffold suchthat the intestinal tissue construct and sphincter construct contacteach other, and the resulting combined sphincter and intestinal tissueconstructs can be further cultured about the scaffold until a unifiedtubular gut-sphincter complex is obtained.

In other aspects, tissue engineered, tubular gut-sphincter complexes aredisclosed for therapeutic inventions or for use as screening or testingplatforms, e.g., for organ on a chip purposes, to test the effects ofdrugs or other therapies on functional gut tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are photographs of an engineered sphincter-rectal cuffcomplex construction. FIG. 1A is a photograph of the complexpre-implantation. FIG. 1B is a photograph of the complex constructionpost-implantation. FIG. 1C is a photograph of an end view of theconstruction, showing maintenance of luminal patency.

FIG. 2A is a graph of changes in basal tone over time of a an engineeredsphincter-rectal cuff complex construction according to the inventionduring an induced contraction.

FIG. 2B is a photograph of the sphincter-rectal cuff complex with thesphincter component delineated.

FIGS. 3A-3D are further photographs of a sphincter-rectal cuff complex.FIG. 3A is a photograph of an engineered sphincter-rectal cuff complexconstruction preimplantation. FIG. 3B is a photograph of an engineeredsphincter-rectal cuff complex construction during implantation. FIG. 3Cis a photograph of an engineered sphincter-rectal cuff complexconstruction immediately following implantation. FIG. 3D is a photographof an engineered sphincter-rectal cuff complex construction at harvestafter 14 days in the animal's abdominal cavity.

FIG. 4 is another photograph of a sphincter-rectal cuff complexfollowing implantation.

FIGS. 5A-5C are photographs of a gut-sphincter complex according to theinvention. FIG. 5A is a photograph that shows the complexpre-implantation, having a gut part 3 cm in length and a sphincter part(identified by a white arrow). FIG. 5B is a photograph that shows thecomplex after 4 weeks of subcutaneous implantation in the abdominal walland shows vascularization of the complex. FIG. 5C is a photograph thatshows that the complex maintained its luminal patency (0.3 cm internaldiameter).

FIG. 6A is a photograph of a gut-sphincter complex measured forintraluminal pressures. FIG. 6B is a schematic illustration of theintraluminal pressure measurement device and system used in FIG. 6A.FIG. 6C is a graph of in vivo intraluminal pressure measurements. Therats were anesthetized and the complex was accessed. A catheter with 4circumferential sensors was inserted into the complex to measure luminalpressure. The catheter was connected to Sandhill equipment that recordsthe pressures (as shown in the diagram for each sensor). Mean gutluminal pressure was 21±2 mmHg while the sphincteric pressure was 52±3mmHg.

FIGS. 7A-7C are graphs showing the mechanical properties of theimplanted complex. The tensile properties of the complex were lower thanthose of the native rat intestine; however they were not significantlydifferent. FIG. 7A is a graph that shows that the tensile strength ofthe complex was 0.043±0.007 MPa compared to 0.067±0.006 MPa for thenative rat intestine. FIG. 7B is a graph that shows that the elongationat break of the implant was 171±30% compared to 230±13% for the nativerat intestine. FIG. 7C is a graph that shows that Young's modulus of theimplanted complex was 0.1±0.01 MPa compared to 0.12±0.01 MPa for thenative rat intestine.

FIGS. 8A and 8B provide histological evaluations of the implants. FIG.8A is an image of H&E for the sphincter. FIG. 8B is an image of H&E forthe gut parts of the complex, showing maintenance of the circularalignment of the smooth muscle around the lumen of the tubular graft.

FIGS. 9A-9F are photographs showing the immunofluorescence of theimplants. FIG. 9A shows the smooth muscle of the sphincter. FIG. 9Bshows the smooth muscle of the gut parts. Together, FIG. 9A and FIG. 9Bshow that the sphincter and gut parts of the complex maintained theircontractile phenotype as shown by positive stain for smoothelin after 4weeks of implantation. FIG. 9C demonstrates the innervation of thecomplex as demonstrated by positive stain with βIII tubulin, indicatingthat the neural progenitor cells differentiated into neurons. FIG. 9Dshows the presence of excitatory motor neurons, demonstrated by positivestain with ChAT. FIG. 9E shows inhibitory motor neurons stained positivewith nNOS. FIG. 9F demonstrates vascularization by positive stain withvon Willebrand factor.

FIG. 10A-10C are graphs showing in vitro physiological functionality ofthe implants. FIG. 10A shows that the implanted sphincter maintained itscapacity to generate a spontaneous basal tone of 382±79 μN. FIG. 10Bshows that the addition of potassium chloride (KCl) resulted in acontraction of 427±42 μN above the basal tone in the sphincter. FIG. 10Cshows that KCl resulted in a robust and sustained contraction (434±17μN) in the gut part of the complex.

FIGS. 11A-11D are further graphs showing in vitro physiologicalfunctionality of the implants. FIG. 11A is a graph that shows electricalfield stimulation (EFS) of the sphincter. FIG. 11B is a graph that alsoshows electrical field stimulation (EFS) of the sphincter. FIG. 11C is agraph that shows electrical field stimulation of the gut parts of thecomplex. Together FIGS. 11A-11C show that electrical field stimulationcaused relaxation of the smooth muscle (black trace). FIG. 11D is agraph that shows that pre-incubation of the implants with nNOS inhibitorLNAME significantly reduced the magnitude of relaxation, indicating thepresence of functional nitrergic neurons (See also the grey traces ofFIGS. 11B & 11C).

DETAILED DESCRIPTION

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. The steps of any method canbe practiced in feasible order and are restricted to a sequential ordermerely because they are so recited in a claim.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

“Differentiation” refers to a change that occurs in cells to cause thosecells to assume certain specialized functions and to lose the ability tochange into certain other specialized functional units. Cells capable ofdifferentiation may be any of totipotent, pluripotent or multipotentcells. Differentiation may be partial or complete with respect to matureadult cells.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts. Neural stem cellscan be isolated from embryonic and adult central nervous system (CNS)tissue, neural tube tissue or enteric nervous system (ENS) tissue.

Stem cells can be further classified according to their developmentalpotential as: (1) totipotent; (2) pluripotent; (3) multipotent; (4)oligopotent; and (5) unipotent. Totipotent cells are able to give riseto all embryonic and extra-embryonic cell types. Pluripotent cells areable to give rise to all embryonic cell types. Multipotent cells includethose able to give rise to a subset of cell lineages, but all within aparticular tissue, organ, or physiological system (for example,hematopoietic stem cells (HSC) can produce progeny that include HSC(self-renewal), blood cell-restricted oligopotent progenitors, and allcell types and elements (e.g., platelets) that are normal components ofthe blood). Cells that are oligopotent can give rise to a morerestricted subset of cell lineages than multipotent stem cells; andcells that are unipotent typically are only able to give rise to asingle cell lineage.

In a broader sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase “differentiates into a neural lineage orphenotype” refers to a cell that becomes partially or fully committed toa specific neural phenotype of the CNS or PNS, i.e., a neuron or a glialcell, the latter category including without limitation astrocytes,oligodendrocytes, Schwann cells and microglia. The term “neural” as usedherein is intended to encompass all electrical active cells, e.g., cellsthat can process or transmit information through electrical or chemicalsignals, including the aforementioned neurons, glial cells, astrocytes,oligodendrocytes, Schwann cells and microglia.

For the purposes of this disclosure, the terms “neural progenitor cell”or “neural precursor cell” mean a cell that can generate progeny thatare either neuronal cells (such as neuronal precursors or matureneurons) or glial cells (such as glial precursors, mature astrocytes, ormature oligodendrocytes). Typically, the cells express some of thephenotypic markers that are characteristic of the neural lineage.Typically, they do not produce progeny of other embryonic germ layerswhen cultured by themselves in vitro, unless dedifferentiated orreprogrammed in some fashion.

A “neuronal progenitor cell” or “neuronal precursor cell” is a cell thatcan generate progeny that are mature neurons. These cells may or may notalso have the capability to generate glial cells. A “glial progenitorcell” or “glial precursor cell” is a cell that can generate progeny thatare mature astrocytes or mature oligodendrocytes. These cells may or maynot also have the capability to generate neuronal cells.

The phrase “biocompatible substance” and the terms “biomaterial” and“substrate” are used interchangeably and refer to a material that issuitable for implantation or injection into a subject. A biocompatiblesubstance does not cause toxic or injurious effects once implanted inthe subject. In one embodiment, the biocompatible substrate includes atleast one component of extracellular matrix. In other embodiments, thesubstrate can also include a polymer with a surface that can be shapedinto the desired structure that requires repairing or replacing. Thepolymer can also be shaped into a part of a body structure that requiresrepairing or replacing. In another embodiment, the biocompatiblesubstrate can be injected into a subject at a target site.

In one embodiment, the substrate is an injectable or implantablebiomaterial that can be composed of crosslinked polymer networks whichare typically insoluble or poorly soluble in water, but can swell to anequilibrium size in the presence of excess water. For example, ahydrogel can be injected into desired locations within the organ. In oneembodiment, the collagen can be injected alone. In another embodiment,the collagen can be injected with other hydrogels. The hydrogelcompositions can include, without limitation, for example, poly(esters),poly(hydroxy acids), poly(lactones), poly(amides), poly(ester-amides),poly(amino acids), poly(anhydrides), poly(ortho-esters),poly(carbonates), poly(phosphazines), poly(thioesters), polysaccharidesand mixtures thereof. Furthermore, the compositions can also include,for example, a poly(hydroxy) acid including poly(alpha-hydroxy) acidsand poly(beta-hydroxy) acids. Such poly(hydroxy) acids include, forexample, polylactic acid, polyglycolic acid, polycaproic acid,polybutyric acid, polyvaleric acid, and copolymers and mixtures thereof.

Hydrogels with effective pore sizes in the 10-100 nm range and in the100 nm-10 micrometer range are termed “microporous” and “macroporous”hydrogels, respectively. Microporous and macroporous hydrogels are oftencalled polymer “sponges.” When a monomer, e.g., hydroxyethylmethacrylate (HEMA), is polymerized at an initial monomer concentrationof 45 (w/w) % or higher in water, a hydrogel is produced with a porosityhigher than the homogeneous hydrogels. Hydrogels can also expand in thepresence of diluent (usually water). The matrix materials of presentinvention encompass both conventional foam or sponge materials and theso-called “hydrogel sponges.” For a further description of hydrogels,see U.S. Pat. No. 5,451,613 (issued to Smith et al.) herein incorporatedby reference.

The term “extracellular matrix” or “ECM” is used herein to denotecompositions comprising one or more of the following: collagen I,collagen IV, laminin, heparan sulfate, or fragments of one or more ofsuch proteins.

“Collagen I” refers to collagen I or collagen I compositions derivedfrom cell culture, animal tissue, or recombinant means, and may bederived from human, murine, porcine, or bovine sources. “Collagen I”also refers to substances or polypeptide(s) at least substantiallyhomologous to collagen I or collagen I compositions. Additionally,“collagen I” refers to collagen I or collagen I compositions that do notinclude a collagen I fragment, e.g., including essentially only acomplete collagen I protein.

“Collagen IV” refers to collagen IV or collagen IV compositions derivedfrom cell culture, animal tissue, or recombinant means, and may bederived from human, murine, porcine, or bovine sources. “Collagen IV”also refers to substances or polypeptide(s) at least substantiallyhomologous to collagen IV or collagen IV compositions. Additionally,“collagen IV” refers to collagen IV or collagen IV compositions that donot include a collagen IV fragment, e.g., including essentially only acomplete collagen I protein.

“Laminin” refers to laminin, laminin fragments, laminin derivatives,laminin analogs, or laminin compositions derived from cell culture,recombinant means, or animal tissue. “Laminin” can be derived fromhuman, murine, porcine, or bovine sources. “Laminin” refers to lamininor laminin compositions comprising laminin-1, laminin-2, laminin-4, orcombinations thereof. “Laminin” also refers to substances orpolypeptide(s) at least substantially homologous to laminin-1,laminin-2, or laminin-4. Additionally, “laminin” refers to laminin orlaminin compositions that do not include a laminin fragment, e.g.,including essentially only a complete laminin protein.

The term “subject” as used herein refers to any living organism,including, but not limited to, humans, nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats, rabbitsand guinea pigs, and the like. The term does not denote a particular ageor sex. In a specific embodiment, the subject is human.

The terms “treating,” “treatment” or “intervention” refer to theadministration of one or more therapeutic agents or procedures to asubject who has a condition or disorder or a predisposition toward acondition or disorder, with the purpose to prevent, alleviate, relieve,alter, remedy, ameliorate, improve, affect, slow or stop theprogression, slow or stop the worsening of the disease, at least onesymptom of condition or disorder, or the predisposition toward thecondition or disorder.

The gut is responsible for ingestion of food, propulsion of luminalcontent and excretion of waste. These functions are conducted by thebasic unit of the musculature of the gut, which is the smooth muscle.Function of the smooth muscle of the gut is highly regulated, mainly bythe enteric nervous system (ENS) and the interstitial cells of Cajal(ICCs). The smooth muscle receives regulatory inputs which are criticalto produce a coordinated response. While the gut is considered acontinuous tubular muscular organ (except for the stomach), severalsphincters exist as checkpoints along the length of the gut. Thosesphincters possess high pressure zone that regulate the propulsion ofluminal content. Neuro-muscular diseases of the gut alter the normalmotility patterns. Even though surgical intervention remains thestandard treatment, preservation of the sphincter attached to the restof the gut is challenging.

Motility disorders result when neuro-muscular functions of the gut aredisturbed. In certain cases of neuro-muscular diseases, sphincterintegrity and function is also impaired. In esophageal achalasia,impairment of the enteric neurons at the level of the smooth muscle ofthe lower esophagus causes loss of relaxation of the lower esophagealsphincter (LES). Treatments aim to reduce the contractility of the LES.This can be done using different drugs/blockers, balloon dilatation,injection of botulinum toxin or myotomy of the LES. On the other hand,gastroparesis is characterized by delayed gastric emptying. This ispartially attributed to the inability of the pyloric sphincter to relax.Common treatments include botulinum toxin injection, drugs/diet change,gastric electric stimulation or pyloroplasty. In patients withcolorectal cancer, preservation of the sphincter following surgicalresection of the tumor is challenging. The formation of a stoma is acommon treatment; however patients suffer from low quality of life(social and physical problems). In other cases, congenital anomaliessuch as anorectal malformation involve the rectum along with the anus.Children with anorectal malformation require surgical intervention whichconsists of either surgical repair or a colostomy. Fecal incontinence isa major complication among other physical and social morbiditiesresulting from anorectal malformation. All of the listed treatments forneuro-muscular disorders are either associated with complications orprovide a short-term relief for the patients. New long term therapeuticstrategies are needed.

Methods and constructs are disclosed for bioengineering ofgastrointestinal tissue. In particular, three-dimensional,bioengineered, tubular gut-sphincter complexes (TGSC) are disclosed,together with methods of forming such constructs. Specifically,engineered innervated human smooth muscle sheets and innervated humansphincters with a pre-defined alignment are disclosed for placementaround tubular scaffolds to create a gut-sphincter complex.

In certain embodiments, a method of forming a tissue engineered, tubulargut-sphincter complex comprises: isolating intestinal circular smoothmuscle cells from an intestinal donor source, isolating sphinctericsmooth muscle cells from a sphincteric donor source, isolating entericneural progenitor cells from at least one neural progenitor donorsource, seeding the isolated intestinal circular smooth muscle cells ona mold with a surface texture that induces longitudinal alignment of theintestinal circular smooth muscle cells, adding the isolated entericneural progenitor cells to the intestinal circular smooth muscle cellson the mold, co-culturing the intestinal circular smooth muscle cellsand the enteric neural progenitor cells until an innervated alignedsmooth muscle sheet is obtained, disposing the innervated aligned smoothmuscle sheet around a tubular scaffold to form an innervated intestinaltissue construct, admixing the sphincteric smooth muscle cells andadditional enteric neural progenitor cells in a biocompatiable gelsolution, applying the gel and admixed cells to a mold having a centralpost; co-culturing the sphinteric smooth muscle cells and neuralprogenitor cells to form an innervated sphincter construct around themold post, transferring and applying the innervated sphincter constructto the tubular scaffold such that the innervated intestinal tissueconstruct and the innervated sphincter construct contact each other, andfurther culturing the combined sphincter and intestinal tissueconstructs about the scaffold until a unified tubular gut-sphinctercomplex is obtained.

In embodiments described herein, the steps of isolating the intestinalcircular smooth muscle cells, sphincteric smooth muscle cells andenteric neural progenitor cells may further comprise obtaining each typeof cell from a single subject. In some embodiments, the intestinalcircular smooth muscle cells, sphincteric smooth muscle cells andenteric neural progenitor cells are obtained from a subject who isbetween the ages of about 1 year to about 80 years.

In embodiments described herein, at least one co-culturing step maycomprise culturing cells in a collagen suspension.

In embodiments described herein, the step of disposing the innervatedaligned smooth muscle sheet around a tubular scaffold may comprisewrapping the innervated aligned smooth muscle sheet around a chitosanscaffold.

In embodiments described herein, the method of forming a tissueengineered, tubular gut-sphincter complex may further compriseconnecting two or more innervated aligned smooth muscle sheets togetherto form a composite structure.

In some embodiments, the unified tubular gut-sphincter complex has alength of about 1 cm to about 100 cm. In certain embodiments, theunified tubular gut-sphincter complex has a width of about 0.1 cm toabout 10 cm.

In certain embodiments, a tubular gut-sphincter complex is formed byisolating intestinal circular smooth muscle cells from an intestinaldonor source, isolating sphincteric smooth muscle cells from asphincteric donor source, isolating enteric neural progenitor cells fromat least one neural progenitor donor source, seeding the isolatedintestinal circular smooth muscle cells on a mold with a surface texturethat induces longitudinal alignment of the intestinal circular smoothmuscle cells, adding the isolated enteric neural progenitor cells to theintestinal circular smooth muscle cells on the mold, co-culturing theintestinal circular smooth muscle cells and the enteric neuralprogenitor cells until an innervated aligned smooth muscle sheet isobtained, disposing the innervated aligned smooth muscle sheet around atubular scaffold to form an innervated intestinal tissue construct,admixing the sphincteric smooth muscle cells and additional entericneural progenitor cells in a biocompatiable gel solution, applying thegel and admixed cells to a mold having a central post; co-culturing thesphinteric smooth muscle cells and neural progenitor cells to form aninnervated sphincter construct around the mold post, transferring andapplying the innervated sphincter construct to the tubular scaffold suchthat the innervated intestinal tissue construct and the innervatedsphincter construct contact each other, and further culturing thecombined sphincter and intestinal tissue constructs about the scaffolduntil a unified tubular gut-sphincter complex is obtained.

In embodiments described herein, steps of isolating the intestinalcircular smooth muscle cells, sphincteric smooth muscle cells andenteric neural progenitor cells in the formation of the tubulargut-sphincter complex may further comprise obtaining each type of cellfrom a single subject. In some embodiments, the intestinal circularsmooth muscle cells, sphincteric smooth muscle cells and enteric neuralprogenitor cells are obtained from a subject who is between the ages ofabout 1 year to about 80 years.

In embodiments described herein, in the formation of the tubulargut-sphincter complex at least one co-culturing step may compriseculturing cells in a collagen suspension.

In embodiments described herein, in the formation of the tubulargut-sphincter complex the step of disposing the innervated alignedsmooth muscle sheet around a tubular scaffold may comprise wrapping theinnervated aligned smooth muscle sheet around a chitosan scaffold.

In embodiments described herein, the formation of a tissue engineered,tubular gut-sphincter complex may further comprise connecting two ormore innervated aligned smooth muscle sheets together to form acomposite structure.

In some embodiments, the tubular gut-sphincter complex has a length ofabout 1 cm to about 100 cm. In certain embodiments, the unified tubulargut-sphincter complex has a width of about 0.1 cm to about 10 cm.

In certain embodiments, a tubular gut-sphincter complex comprises: atubular scaffold; an innervated intestinal tissue construct disposedabout the tubular scaffold; and an innervated sphincteric tissueconstruct also disposed about the tubular scaffold joined to theintestinal tissue construct at an end of the intestinal tissueconstruct; and the complex exhibits directionally oriented smooth musclecells, basal tone and choleric contractions in response to a contractilestimulus.

In embodiments described herein, the tubular scaffold may furthercomprise a chitosan scaffold.

In some embodiments, the tubular gut-sphincter complex has a length ofabout 1 cm to about 100 cm. In certain embodiments, the unified tubulargut-sphincter complex has a width of about 0.1 cm to about 10 cm.

To demonstrate the utility of the present invention, engineeredcomplexes were subcutaneously implanted in the abdomen of the rats for 4weeks. The implanted tissues exhibited vascularization and in vivomanometry revealed luminal pressure at the gut and the sphincter zone.Tensile strength, elongation at break and Young's modulus of theengineered complexes were similar to those of native rat intestine.Histological and immunofluorescence assays showed maintenance of smoothmuscle circular alignment in the engineered tissue, maintenance ofsmooth muscle contractile phenotype and innervation of the smoothmuscle. Electrical field stimulation induced a relaxation of the smoothmuscle of both the sphincter and the gut parts. Relaxation was partiallyinhibited by nitric oxide inhibitor indicating nitrergic contribution tothe relaxation. The sphincteric part of TGSC maintained the basal tonecharacteristic of a native sphincter. The gut part also maintained itsspecific neuro-muscular characteristics. The results of this studyprovide a promising therapeutic approach to restore gut continuity andmotility.

EXAMPLe 1 Spincter-Rectal Cuff Complex

Innervated aligned smooth muscle sheets were engineered using theco-culture technique of isolated human smooth muscle cells and humanenteric neural progenitor cells derived from the small intestine, asdescribed above. The sheets were wrapped around tubular chitosanscaffolds. Innervated pyloric sphincters were engineered using humanpyloric smooth muscle and enteric neural progenitor cells, again asdescribed above. The sphincters were placed on one end around thetubular scaffold. The combination of the tissues around the tubularscaffold is referred to as sphincter-rectal cuff. The sphincter-rectalcuff was 3 cm in length and 3 mm internal diameter.

Athymic rats were used for this study to avoid implant rejection sincethe implant was made of human cells. Following sedation of the rats, a 5cm midline skin incision was made. The engineered tubular tissue wasimplanted subcutaneously and fixed in place using non-resorbablesutures. FIG. 5A is a photograph of bioengineered sphincter-rectal cuffpre-implantation.

Four weeks following implantation, the rats were brought to theprocedure room and anesthetized using isoflurane. A midline incision wasmade and the surgical site was re-accessed. The implantedsphincter-rectal cuff was healthy in color and highly vascularized

The implant maintained its luminal patency for 4 weeks. A manometrycatheter was used to measure the luminal pressure in thesphincter-rectal cuff. The sensors were linearly aligned along thecatheter and were 2 mm apart. The catheter was inserted into the lumenof the sphincter-rectal cuff while still attached under the skin.Luminal pressure was recorded in all 3 channels and averaged 40 mmHg.Spontaneous small amplitude contractions were seen in all 3 channels.The rat was then euthanized and the implant was harvested.

FIGS. 1A-1C are photographs of an engineered sphincter-rectal cuffcomplex construction. FIG. 1A shows the complex pre-implantation whileFIG. 1B shows the complex construction post-implantation and FIG. 1C isan end view the construction, showing maintenance of luminal patency.FIG. 2A is a graph of changes in basal tone over time during an inducedcontraction. FIG. 2B is a photograph of the sphincter-rectal cuffcomplex with the sphincter component delineated. FIGS. 3A-3D are furtherphotographs of a sphincter-rectal cuff complex preimplantation (FIG.3A), during implantation (FIG. 3B), immediately following implantation(FIG. 3C) and at harvest after 14 days in the animals abdominal cavity(FIG. 3D). FIG. 4 is another photograph of sphincter-rectal cuff complexfollowing implantation.

Following measurement of luminal pressure, one end of thesphincter-rectal cuff was tightly clamped while the other end was leftopen. A volume of 1 ml of liquid was pipetted through the open end. Thetissue expanded in the center as it was filled with liquid. The tissuethen returned to its original shape after the liquid solution wascleared. There was no sign of leakage or disruption.

Cross sections of the sphincter and the rectal cuff were used forphysiological studies. The sphincter maintained its ability to generatea spontaneous basal tone similar to an engineered sphincter prior to itsimplantation. This indicates that the implanted sphincter maintains itstonic characteristic in vivo. The electromechanical coupling integrityof the smooth muscle was also evaluated in the presence of potassiumchloride. Following depolarization of the smooth muscle membrane in boththe sphincter and the rectal cuff, a fast and robust contraction wasobserved. This indicates that the voltage-dependent calcium channelswere maintained in the sphincter-rectal cuff after 4 weeks ofimplantation. Next, we evaluated the response of the sphincter-rectalcuff to exogenous neurotransmitters. The addition of acetylcholine (Ach)caused a rapid contraction, which was significantly attenuated when thetissue was pre-treated with the neurotoxin tetrodotoxin (TTX). Thisindicates that Ach acted on both the smooth muscle and the neurons andcaused muscle contraction. After blocking the neurons with TTX, Achacted on smooth muscle receptors only and caused a lower response.Relaxation was evaluated in the presence of VIP (vasoactive intestinalpolypeptide). In the presence of TTX, the observed VIP-inducedrelaxation was significantly diminished. Again, this indicates that VIPrelaxation was mediated through receptors on the smooth muscle andneurons. To further confirm the functionality of the neurons, electricalfield stimulation (EFS) was applied on the implant. EFS caused a rapidrelaxation which was completely inhibited by TTX. This indicated thatthe response was purely neuronally mediated.

Additional cross sections of the sphincter and the rectal cuff werefixed in formalin and processed for histological analysis. H&E stainingshowed maintenance of alignment of smooth muscle around the lumen of thetubular scaffold. Maintenance of smooth muscle phenotype was confirmedby positive immunostaining for smooth muscle specific markers.Innervation was also confirmed by positive satining for neural markerβIII tubulin. Vascularization was further confirmed by staining for vonWillebrand factor.

Cross sections of the implanted tissue were obtained and weremechanically evaluated using a mechanical testing machine (Instron). Thetissues were subjected to tensile stress and strain testing. Young'smodulus of the implant was higher than the scaffold only, indicatingthat the innervated smooth muscle sheet remodeled around the scaffoldfollowing implantation resulting in higher Young's modulus. This is anindication that the implant was able to handle higher stretch withoutbreaking when compared to scaffolds only.

EXAMPLE 2 Sphincter-Intestine Complex

Materials and Methods:

Cell culture reagents were purchased from Life Technologies (GrandIsland, N.Y., US) unless otherwise specified. Smooth muscle growthmedium consisted of Dulbecco's modified Eagle medium, 10% fetal bovineserum, 1.5% antibiotic-antimycotic, and 0.6% L-glutamine. Neural growthmedium consisted of neurobasal, 1×N2 supplement, recombinant humanEpidermal Growth Factor (EGF 20 ng/mL, Stemgent, San Diego, Calif., US),recombinant basic Fibroblast Growth Factor (bFGF 20 ng/mL, Stemgent,Calif., US), and 1×antibiotic-antimycotic. Neural differentiation mediaconsisted of neurobasal medium-A supplemented with 2% fetal bovineserum, 1×B27 supplement and 1×antibiotic-antimycotic. Medium molecularweight chitosan (75-85% deacetylation), tetrodotoxin (TTX), and neuronalnitric oxide synthase (nNOS)-blocker N_(ω)-Nitro-L-arginine methyl esterhydrochloride (L-NAME) were purchased from Sigma (St. Louis, Mo.).Sylgard [poly(dimethylsiloxane); PDMS] was purchased from WorldPrecision Instruments (Sarasota, Fla.). Type I rat tail collagen waspurchased from BD Biosciences.

Human intestinal and pyloric tissues were ethically obtained from organdonors through Carolina Donor Services and Wake Forest Baptist MedicalCenter (IRB No. 00007586). Tissues were obtained from three donors aged2, 18, and 67 years.

Human intestinal circular smooth muscle cells: Smooth muscle cells wereisolated from human duodenum. The duodena (10 cm below the pyloricsphincter) were obtained consistently for cell isolation. Human duodenawere cleaned of any luminal content and were washed extensively inice-cold Hank's balanced salt solution (HBSS). The tissues were cut intosmaller pieces and the circular smooth muscle was obtained by strippingoff the mucosa and the longitudinal muscle. The circular smooth muscletissue was then minced, washed extensively in HBSS and incubated in adigestion mix containing type II collagenase (Worthington, Lakewood,N.J.) and DNAse (Roche, Indianapolis, Ind.) for one hour at 37° C. withagitation. The digested tissue was then extensively washed in HBSS andsubjected to a second digest. Digested cells were washed, resuspended inwarm smooth muscle growth media and expanded in tissue culture flasks at37° C. with 5% CO₂.

Human pyloric smooth muscle cells: Human pylori were dissected off forsmooth muscle isolation. Pylorus tissues were cleaned of any fat andmucosa and extensively washed with HBSS. Tissues were then minced andwashed again with sterile HBSS. Minced tissues were digested twice at37° C. with agitation in type II collagenase (Worthington Biochemical,Lakewood, N.J.) and DNAse (Roche, Indianapolis, Ind.) for one hour each.Cells were pelleted down with centrifugation, resuspended in smoothmuscle growth media and expanded in tissue culture flasks at 37° C. with5% CO₂.

Human enteric neural progenitor cells: Human enteric neural progenitorcells were isolated from the small intestine. Human duodenal tissueswere finely minced followed by extensive washing in HBSS. Tissues werethen digested in a mixture of type II collagenase, dispase, and DNAse.The cells were passed through 70 μm cell strainer followed by extensivewashing. The cells were then passed through 40 μm cell strainers.Following centrifugation, cells were resuspended in neural growth mediaand cultured in non-tissue culture treated plates at 37° C. and 7% CO₂.The cultured cells formed free-floating clusters referred to asneurospheres which have been shown to stain positive for neuralcrest-derived cell marker p75.

Preparation of tubular gut-sphincter segment: Tubular chitosan/collagenscaffolds were engineered as described by Zakhem et al. in“Chitosan-based scaffolds for the support of smooth muscle constructs inintestinal tissue engineering,” Biomaterial, 2012; 33:4810-7 and Zakhemet al. in “Development of Chitosan Scaffolds with Enhanced MechanicalProperties for Intestinal Tissue Engineering Applications,” Journal ofFunctional Biomaterials, 2015; 6:999-1011. A 2% w/v chitosan solutionwas prepared in 0.2 M acetic acid. The chitosan solution was then mixedwith type I collagen in a 1:1 ratio. The mix was then poured into acustom-made 3-cm long tubular mold with a diameter of 0.7 cm. The lumenof the scaffold was created by inserting an inner tubing of 0.3 cmdiameter in the center of the main tubular mold. This created a scaffoldwith length of 3 cm and internal diameter of 0.3 cm. The scaffolds werefrozen at −80° C. for 3 hours followed by lyophilization overnight. Thescaffolds were then neutralized in 0.2 NaOH and washed extensively withPBS and distilled water. The scaffolds were sterilized in 70% ethanoland then washed extensively with sterile 1× PBS before cell seeding.

Innervated aligned smooth muscle sheets were engineered as previouslydescribed by Zakhem et al. in “Successful implantation of an engineeredtubular neuromuscular tissue composed of human cells and chitosanscaffold,” Surgery, 2015; 158:1598-608. and Zakhem et al. in“Development of Chitosan Scaffolds with Enhanced Mechanical Propertiesfor Intestinal Tissue Engineering Applications,” Journal of FunctionalBiomaterials, 2015; 6:999-1011. Briefly, smooth muscle cells were seededonto wavy molds made of Sylgard with longitudinal grooves and allowed toalign. Five days after smooth muscle alignment, neural progenitor cellswere collected and suspended in a mixture of 10% FBS, 1× DMEM, 1×antibiotic-antimycotic, 10 m/ml mouse laminin and 0.4 mg/ml type I rattail collagen. Neural progenitor cells were then mixed incollagen/laminin gel and laid on top of the smooth muscle. Engineeredhuman innervated smooth muscle sheets were then circumferentiallywrapped around the tubular scaffolds as described previously to mimicthe circular muscle layer. The sheets around the scaffolds are referredto as the gut part of the complex. Human innervated pyloric smoothmuscle sphincters were engineered as previously described by Rego et al.in “Bioengineered human pyloric sphincters using autologous smoothmuscle and neural progenitor cells,” Tissue engineering, 2015. Asuspension of 200 000 enteric neural progenitor cells was suspended in agel mix. The mixture was pipetted on a Sylgard-coated plate that had acentral cylindrical post and allowed to gel for about 20 min at 37° C.Pyloric smooth muscle cells were trypsinized and 500 000 cells wereobtained. The cells were resuspended in a similar gel mixture. Themixture was then pipetted on top of the first neural layer. Followinggelation, differentiation media was supplemented every other day for 10days. The sphincters were also placed, at one end, around the tubularscaffolds and were referred to as the sphincter part of the complex.

Implantation of the engineered tubular gut-sphincter complex: Athymicrats (n=6) were used as recipients of the tubular gut-sphinctercomplexes. Surgical procedures described in this work were performedfollowing the guidelines set forth by IACUC. Rats were anesthetized bycontinuous isoflurane masking throughout the surgery. The surgical areawas shaved and aseptically prepared. A midline skin incision of up to 5cm was made in the abdominal wall. The engineered tubular gut-sphinctercomplex was fixed using 5-0 prolene sutures to mark the tissue at thetime of harvest. The rats were allowed to recover in their cages instandard fashion and were given the appropriate analgesics.

In vivo intraluminal pressure measurement: Four weeks followingimplantation, the rats were brought back to the procedure room. The ratswere anesthetized by continuous isoflurane masking. The surgical sitewas re-accessed. The implants were located by the prolene sutures. Anair-charged catheter with circumferential sensors (7 mm spacing betweenthe sensors) was used to measure the luminal pressure of the tubularimplants. The catheter was inserted into the tubular implant enteringfrom the gut part until the first sensor of the catheter reached thesphincteric area. The remaining sensors measured the pressure at the gutpart of the tubular implant. Luminal pressure was recorded using InSIGHTAcquisition (version 5.2.4, Sandhill scientific Inc, Highland Ranch,Colo., USA). The recorded pressures were analyzed using BioVIEW system(version 5.6.3.0 Sandhill scientific Inc, Highland Ranch, Colo., USA).Following pressure readings, the rats were euthanized. The implants weredissected from the surrounding tissue. The harvested implants werefurther evaluated.

Immediately after harvest, the implants were tested for their tensileproperties using a uniaxial load test machine (Instron model #5544,Issaquah, Wash., USA). Tubular specimens were obtained and hooked ontothe machine equipped with a 2 kN load cell. Tensile strength, elongationat break and Young's modulus were obtained. Rat native intestines servedas control.

A pressure transducer catheter with an inflatable balloon was used tomeasure the burst strength pressure of the implants. The catheter wasinserted into the lumen of the implants and the luminal pressure wasincreased until failure occurred. The pressure was slowly increaseduntil failure occurred and the pressures were recorded.

Immediately after harvest, sections of the sphincter and the gut tissueswere fixed in formaldehyde, processed and paraffin embedded. Sectionswere deparaffinized and stained with hematoxylin and eosin (H&E) formorphological analysis. Phenotype of smooth muscle and differentiatedneurons was analyzed by incubating the sections in primary antibodiesdirected against smoothelin and β-III tubulin, respectively. Neuronalnitric oxide synthase (nNOS) and choline acetyltransferase (ChAT)antibodies were used to confirm the presence of inhibitory andexcitatory motor neurons, respectively. Vascularization was confirmed byimmunostaining of the sections with von Willebrand (vWF) factor.Appropriate fluorophore-conjugated secondary antibodies were used.

Circular strips of the harvested sphincters and gut tissues were alsoimmediately obtained and evaluated for physiological functionality. Aforce transducer apparatus (Harvard Apparatus, Holliston, Mass.) wasused to measure real time force generation. The tissues were hooked to astationary fixed pin from one side and to the measuring arm of the forcetransducer from the other side. The tissues were kept in a warm tissuebath throughout the experiments. Establishment of basal tone by thesphincters, electromechanical coupling integrity of the smooth muscle ofboth the sphincters and the gut tissues, and the functionality of thedifferentiated neurons were evaluated.

Electromechanical coupling integrity was evaluated in the presence of 60mM potassium chloride (KCl). Functionality of neurons was evaluatedusing electrical field stimulation (EFS) in the absence and presence ofneurotoxin, tetrodotoxin (TTX) and nitric oxide synthase (nNOS) blockerN_(ω)-Nitro-L-arginine methyl ester hydrochloride (L-NAME).

The difference in tensile strength, elongation at break and Young'smodulus between the implants and the native rat intestines was evaluatedby Student's t-test. Analysis of acquired force data was acquired usingPowerlab and exported to GraphPad Prism 5.0 for Windows (GraphPadSoftware, San Diego, Calif.; www.graphpad.com). Second orderSavitzky-Golay smoothing was applied to data. Student paired t-test wasused to compare the means of forces in the absence and presence ofinhibitors. All values were expressed as means±SEM. A p-value less than0.05 was considered significant.

Results:

Gut-sphincter complex were engineered by combining innervated smoothmuscle sheets and engineered innervated pyloric sphincters aroundtubular chitosan scaffolds (FIG. 5A). The engineered innervated smoothmuscle sheets were wrapped circumferentially around the tubularscaffolds to form the circular muscle layer. The sheets constitute thegut part of the gut-sphincter complex. The engineered sphincters of thecomplex were placed at one end of the scaffolds. The bioengineeredtissues were implanted subcutaneously in the abdomen of athymic rats for4 weeks. At the end of 4 weeks implantation, the tissue engineeredsphincter became integrated with the gut sphincter complex and formed asingle continuous functional unit. The implants showed healthy pinkcolor upon harvest (FIG. 5B). The implants were 3 cm in length and 0.5cm diameter (FIG. 5C). The luminal patency of the implants wasmaintained for 4 weeks post-implantation. There were no signs ofinflammation, infection or tissue necrosis. Neovascularization wasvisually demonstrated by the presence of blood vessels around theimplants.

The rats were anesthetized by continuous isoflurane masking. Theimplants were re-accessed. The catheter with circumferential sensors wascalibrated before any measurement. The pressure reading was set to zeroprior to insertion of the catheter into the lumen of the implant. Thecatheter was inserted into the lumen of the implant by increment of 1sensor at a time until all sensors were inserted (FIGS. 6A-6C).Pressures started increasing as the catheter was inserted into thetissue. Pressure reading from each sensor was reflected on a separatechannel. The catheter was inserted from the gut side of the implant(opposite end of the sphincter). Pressure reading of the sphincter isshown on the top graph (FIG. 6C). The other lower channels weremeasuring the gut part of the complex (FIG. 6C, lower three graphs). Themean luminal pressure (of all sensors) of the gut zone was (21±2 mmHg).The mean pressure recorded at the sphincter zone was (52±3 mmHg). Thepressures were stable over time. The luminal patency was furtherconfirmed by completely inserting the catheter through the length of theimplant without obstruction.

Uniaxial tensile properties of the implants were compared to those ofnative rat intestine. The tensile strength was significantly differentbetween the implants and the native rat intestine (FIG. 7A). The tensilestrength of the native rat intestine was 0.067±0.006 MPa whereas theaverage tensile strength of the implants was 0.043±0.007 MPa (n=4,p=0.02). Elongation at break (FIG. 7B) and Young's modulus (FIG. 7C) ofthe implants were lower than those of the native rat intestine, however,they were not significantly different (n=4, p=0.1 and p=0.37respectively). Elongation at break and Young's modulus were 230±13% and0.12±0.01 MPa, respectively for the native rat intestine. The implant'selongation at break and Young's modulus averaged 171±30% and 0.1±0.01MPa, respectively.

A pressure transducer catheter with an inflatable balloon was insertedinside the lumen of the tubular implants. Pressure was increasedgradually until failure of the implant. The pressure at failure wasrecorded as the burst pressure strength. The mean burst pressurestrength of the implants was 1396±60 mmHg.

Paraffin cross sections of the harvested engineered sphincters and guttissues of 6 μm thickness were prepared. Representative H&E staining ofboth the engineered sphincters and gut tissues after implantation isshown in FIGS. 8A and 8B, respectively. The engineered innervated smoothmuscle sheets were wrapped circumferentially around the tubularscaffolds to form circular muscle layer. The smooth muscle of theengineered sphincters was also circumferentially aligned. H&E stainsshowed maintenance of smooth muscle alignment around the lumen of thetubular tissues for both the gut segment and the sphincter. H&E showsdense aligned smooth muscle.

Sections of both the engineered sphincters and the engineered gutsegments stained positive for the smooth muscle specific markersmoothelin (FIGS. 9A & 9B, respectively). This indicated that smoothmuscle contractile phenotype of both the engineered sphincter and thegut segment was maintained over a period of 4 weeks post-implantation.Engineered tissues also stained positive for pan-neuronal marker,βIII-tubulin, indicating the presence of differentiated neurons in theengineered complex 4 weeks post-implantation (FIG. 9C). Additionalpositive staining for neural nitric oxide synthase (nNOS) (FIG. 9D) andcholine acetyltransferase (ChAT) (FIG. 9E) indicate the presence ofinhibitory and excitatory motor neurons in the implanted complex.Positive stain for Von Willebrand Factor (vWF) confirmed thevascularization of the implants (FIG. 9F).

Circular strips of implanted engineered sphincter and gut segments werehooked to a force transducer measuring arm and allowed to establishbaseline. The engineered sphincters exhibited the spontaneous ability togenerate basal tone that averaged 382±79 μN (FIG. 10A).Electromechanical coupling integrity of both the sphincters and the gutsegments was demonstrated by the robust contraction of the tissues afterthe addition of KCl. Sphincters demonstrated a contraction of 427±42 μNabove the basal tone (FIG. 10B) while the gut tissues exhibited a meanpeak contraction of 434±17 μN (FIG. 10C).

Functionality of neurons in the segment was evaluated by electricalfield stimulation (8 Hz and 0.5 ms). Smooth muscle of both the implantedengineered sphincters and the gut segments relaxed following excitationof the nerves (FIG. 11). Relaxation of the implanted sphincters averaged−294±26 μN below the basal tone (FIGS. 11A and 11B—black traces) and themaximal relaxation averaged −355±8 μN in the gut segment (FIG. 11C—blacktrace). The tissues were then washed with fresh warm buffer andpre-treated with TTX. Upon excitation of the nerves, relaxation wascompletely abolished in both the sphincters and the gut segments. Thisindicated that the relaxation of the smooth muscle observed followingEFS without TTX was due to excitation of neurons only. This alsoindicates that the neural progenitor cells within the complexesdifferentiated into functional neurons. Further characterization of therelaxation response was studied in the presence of nNOS inhibitor LNAME.The tissues were pre-treated with LNAME followed by EFS. Relaxation wassignificantly reduced to −140±6 μN in the sphincters (FIG. 11B—greytrace) and −223±29 in the gut segments (FIG. 11C—grey trace). Thisinhibition indicates that EFS-induced relaxation of the smooth musclewas partially mediated by functional nitrergic neurons. The data fromFIGS. 11B and 11C are quantified in the bar graph depicted in FIG. 11D.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While the preferred embodiments of the invention have been illustratedand described, it will be clear that the invention is not so limited.Numerous modifications, changes, variations, substitutions, andequivalents will occur to those skilled in the art without departingfrom the spirit and scope of the present invention as defined by theappended claims.

The invention claimed is:
 1. A method of forming a tissue engineered,tubular gut-sphincter complex comprising: isolating intestinal circularsmooth muscle cells from an intestinal donor source, isolatingsphincteric smooth muscle cells from a sphincteric donor source,isolating enteric neural progenitor cells from at least one neuralprogenitor donor source, seeding the isolated intestinal circular smoothmuscle cells on a mold with a surface texture that induces longitudinalalignment of the intestinal circular smooth muscle cells, adding theisolated enteric neural progenitor cells to the intestinal circularsmooth muscle cells on the mold, co-culturing the intestinal circularsmooth muscle cells and the enteric neural progenitor cells until aninnervated aligned smooth muscle sheet is obtained, disposing theinnervated aligned smooth muscle sheet around a tubular scaffold to forman innervated intestinal tissue construct, admixing the sphinctericsmooth muscle cells and additional enteric neural progenitor cells in abiocompatible gel solution, applying the gel and admixed cells to a moldhaving a central post; co-culturing the sphincteric smooth muscle cellsand neural progenitor cells to form an innervated sphincter constructaround the mold post, transferring and applying the innervated sphincterconstruct to the tubular scaffold such that the innervated intestinaltissue construct and the innervated sphincter construct contact eachother, and further culturing the combined sphincter and intestinaltissue constructs about the scaffold until a unified tubulargut-sphincter complex is obtained.
 2. The method of claim 1, whereinsteps of isolating the intestinal circular smooth muscle cells,sphincteric smooth muscle cells and enteric neural progenitor cellsfurther comprise obtaining each type of cell from a single subject. 3.The method of claim 1, wherein at least one co-culturing step comprisesculturing cells in a collagen suspension.
 4. The method of claim 1,wherein the step of disposing the innervated aligned smooth muscle sheetaround a tubular scaffold comprises wrapping the innervated alignedsmooth muscle sheet around a chitosan scaffold.
 5. The method of claim1, wherein the method further comprises connecting two or moreinnervated aligned smooth muscle sheets together to form a compositestructure.
 6. A tubular gut-sphincter complex formed by the method ofclaim 1.